MEK Ligands and Polynucleotides Encoding MEK Ligands

ABSTRACT

The invention relates to kinase ligands and polyligands. In particular, the invention relates to ligands, homopolyligands, and heteropolyligands that modulate MEK activity. The ligands and polyligands are utilized as research tools or as therapeutics. The invention includes linkage of the ligands, homopolyligands, and heteropolyligands to a cellular localization signal, epitope tag and/or a reporter. The invention also includes polynucleotides encoding the ligands and polyligands.

This application has subject matter related to application Ser. No.10/724,532 (now U.S. Pat. No. 7,071,295), Ser. No. 10/682,764(US2004/0185556, PCT/US2004/013517, WO2005/040336), Ser. No. 11/233,246,and US20040572011P (WO2005116231). Each of these patents andapplications is hereby incorporated by reference.

FIELD OF INVENTION

The invention relates to mammalian kinase ligands, substrates andmodulators. In particular, the invention relates to polypeptides,polypeptide compositions and polynucleotides that encode polypeptidesthat are ligands, substrates, and/or modulators of MEK. The inventionalso relates to polyligands that are homopolyligands orheteropolyligands that modulate MEK activity. The invention also relatesto ligands and polyligands localized to a subcellular region.

BACKGROUND AND PRIOR ART

Kinases are enzymes that catalyze the addition of phosphate to amolecule. The addition of phosphate by a kinase is calledphosphorylation. When the kinase substrate is a protein molecule, theamino acids commonly phosphorylated are serine, threonine and tyrosine.Phosphatases are enzymes that remove phosphate from a molecule. Theremoval of phosphate is called dephosphorylation. Kinases andphosphatases often represent competing forces within a cell to transmit,attenuate, or otherwise modulate cellular signals and cellular controlmechanisms. Kinases and phosphatases have both overlapping and uniquenatural substrates. Cellular signals and control mechanisms, asregulated by kinases, phosphatases, and their natural substrates are atarget of research tool design and drug design.

MAP/ERK kinase 1, MEK1, PRKMK1, MAPKK1, MAP2K1, MKK1 are the sameenzyme, known as MEK1. MAP/ERK kinase 2, MEK2, PRKMK2, MAPKK2, MAP2K2,MKK2 are the same enzyme, known as MEK2. MEK1 and MEK2 can phosphorylateserine, threonine and tyrosine residues in protein or peptidesubstrates. To date, few cellular substrates of MEK isoforms have beenidentified. While individual substrates or ligands have been identifiedand studied, mixed ligands linked together as polyligands that modulateMEK isoform activity have not been demonstrated before this invention.

Design and synthesis of polypeptide ligands that modulatecalcium/calmodulin-dependent protein kinase and that localize to thecardiac sarco(endo)plasmic reticulum was performed by Ji et al. (J BiolChem (2003) 278:25063-71). Ji et al. accomplished this by generatingexpression constructs that localized calcium/calmodulin-dependentprotein kinase inhibitory polypeptide ligands to the sarcoplasmicreticulum by fusing a sarcoplasmic reticulum localization signal derivedfrom phospholamban to a polypeptide ligand. Sec also U.S. Pat. No.7,071,295.

Detailed Description of Polypeptide and Polynucleotide Sequences

SEQ ID NOS:1-36 are example polyligands and polynucleotides encodingthem.

Specifically, the MEK polyligand of SEQ ID NO:1 is encoded by SEQ IDNO:2, SEQ ID NO:3, and SEQ ID NO:4, wherein the codons have beenoptimized for mammalian expression and vector insertion, and wherein SEQID NOS:3-4 contain alternative flanking restriction sites applicable tomodular cloning methods. SEQ ID NO:1 is an embodiment of a polyligand ofthe structure A-S1-B-S2-C-S3-D, wherein A is SEQ ID NO:41, B is SEQ IDNO:42, C is SEQ ID NO:49, and D is SEQ ID NO:43, wherein Xaa is alanineor phenylalanine, and wherein S1 is a spacer of the amino acid sequencePGAAG, and S2 is a spacer of amino acid sequence PAGGA, and S3 is aspacer of the amino acid sequence PGAAG. A polyligand of structureA-S1-B-S2-C-S3-D is also called herein a heteropolyligand, showngenerically in FIG. 4D.

SEQ ID NO:5 is an embodiment of a polyligand of the structureX-S1-X-S2-Y-S3-Z, wherein X is SEQ ID NO:44, Y is SEQ ID NO:42, Z is SEQID NO:43, wherein Xaa is alanine or phenylalanine, and wherein S1 is aspacer of amino acid sequence PGAAG, S2 is a spacer of the amino acidsequence PAGGA, and S3 is a spacer of the amino acid sequence PGAAG. TheMEK polyligand of SEQ ID NO:5 is encoded by SEQ ID NO:6, SEQ ID NO:7 andby SEQ ID NO:8, wherein the codons have been optimized for mammalianexpression and vector insertion, and wherein SEQ ID NOS:7-8 containalternative flanking restriction sites applicable to modular cloningmethods. A polyligand of structure X-S1-X-S2-Y-S3-Z is also calledherein a heteropolyligand, shown generically in FIG. 4E.

SEQ ID NO:9 is encoded by SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12,wherein the codons have been optimized for mammalian expression andvector insertion, and wherein SEQ ID NOS:11-12 contain alternativeflanking restriction sites applicable to modular cloning methods. SEQ IDNO:1 is an embodiment of a polyligand of the structure A-S1-B-S2-C-S3-D,wherein A is SEQ ID NO:41, B is SEQ ID NO:42, C is SEQ ID NO:49, and Dis SEQ ID NO:43, wherein Xaa is serine, threonine or tyrosine, andwherein S1 is a spacer of the amino acid sequence PGAAG, and S2 is aspacer of amino acid sequence PAGGA, and S3 is a spacer of the aminoacid sequence PGAAG. A polyligand of structure A-S1-B-S2-C-S3-D is alsocalled herein a heteropolyligand, shown generically in FIG. 4D.

SEQ ID NO:13 is an embodiment of a polyligand of the structureX-S1-X-S2-Y-S3-Z, wherein X is SEQ ID NO:44, Y is SEQ ID NO:42, Z is SEQID NO:43, wherein Xaa is serine, threonine or 10 tyrosine, and whereinS1 is a spacer of amino acid sequence PGAAG, S2 is a spacer of the aminoacid sequence PAGGA, and S3 is a spacer of the amino acid sequencePGAAG. The MEK polyligand of SEQ ID NO:13 is encoded by SEQ ID NO:14,SEQ ID NO:15 and by SEQ ID NO:16, wherein the codons have been optimizedfor mammalian expression and vector insertion, and wherein SEQ IDNOS:15-16 contain alternative flanking restriction sites applicable tomodular cloning methods. A polyligand of structure X-S 1-X-S2-Y-S3-Z isalso called herein a heteropolyligand, shown generically in FIG. 4E.

SEQ ID NO:17 is encoded by SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20,wherein the codons have been optimized for mammalian expression andvector insertion, and wherein SEQ ID 20 NOS:19-20 contain alternativeflanking restriction sites applicable to modular cloning methods. SEQ IDNO:17 is an embodiment of a polyligand of the structureA-S1-B-S2-C-S3-D, wherein A is SEQ ID NO:51, B is SEQ ID NO:43, C is SEQID NO:42, and D is SEQ ID NO:44, wherein Xaa is alanine orphenylalanine, and wherein S1 is a spacer of the amino acid sequencePGAAG, and S2 is a spacer of amino acid sequence PAGGA, and S3 is aspacer of the amino acid sequence PGAAG. A polyligand of structureA-S1-B-S2-C-S3-D is also called herein a heteropolyligand, showngenerically in FIG. 4D.

SEQ ID NO:21 is encoded by SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24,wherein the codons have been optimized for mammalian expression andvector insertion, and wherein SEQ ID 30 NOS:23-24 contain alternativeflanking restriction sites applicable to modular cloning methods. SEQ IDNO:21 is an embodiment of a polyligand of the structure A-S1-A-S2-A,wherein A is SEQ ID NO:45, wherein Xaa is alanine or phenylalanine, andwherein S1 is a spacer of the amino acid sequence PGAAG, and S2 is aspacer of amino acid sequence PAGGA, and S3 is a spacer of the aminoacid sequence PGAAG. A polyligand of structure A-S1-A-S2-A is alsocalled herein a homopolyligand, shown generically in FIG. 2D.

SEQ ID NO:25 is encoded by SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28,wherein the codons have been optimized for mammalian expression andvector insertion, and wherein SEQ ID NOS:27-28 contain alternativeflanking restriction sites applicable to modular cloning methods. SEQ IDNO:25 is an embodiment of a monomeric ligand, wherein Xaa is alanine orphenylalanine.

SEQ ID NO:29 is encoded by SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32,wherein the codons have been optimized for mammalian expression andvector insertion, and wherein SEQ ID NOS:31-32 contain alternativeflanking restriction sites applicable to modular cloning methods. SEQ IDNO:29 is an embodiment of a monomeric ligand, wherein Xaa is alanine.

SEQ ID NO:33 is encoded by SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36,wherein the codons have been optimized for mammalian expression andvector insertion, and wherein SEQ ID NOS:35-36 contain alternativeflanking restriction sites applicable to modular cloning methods. SEQ IDNO:33 is an embodiment of a polyligand of the structureA-S4-B-S5-A-S4-B, wherein A is SEQ ID NO:48, B is SEQ ID NO:50, whereinXaa is alanine, and wherein S4 is a spacer of the amino acid sequenceRRPAAA, and S5 is a spacer of amino acid sequence PGGG. A polyligand ofstructure A-S4B-S5-A-S4-B is also called herein a heteropolyligand,shown generically in FIG. 4C.

SEQ ID NOS:37-40 are full length MEK protein substrates or inhibitors.Since MEK undergoes autophosphorylation, MEK is included as a substrate.These sequences have the following public database accession numbers:NP_(—)002746, NP_(—)002737, XP_(—)055766, NP_(—)002736, NP_(—)001744.Each of the sequences represented by these accession numbers isincorporated by reference herein. In SEQ ID NOS:37-40, the positions ofthe amino acid(s) phosphorylatable by MEK are represented by Xaa. Inwild-type proteins, Xaa is serine, threonine, or tyrosine. In theligands of the invention, Xaa is any amino acid.

SEQ ID NOS:41-48 are partial sequences of SEQ ID NOS:37-39, whichrepresent examples of sequences comprising kinase active site blockerpeptide ligand sequences where the location of the MEK phosphorylatableserine, tyrosine, or threonine in the natural polypeptide is designatedas Xaa.

SEQ ID NOS:49-51 are partial sequences of SEQ ID NO:38 or SEQ ID NO:40,which represent examples of peptide kinase inhibitors. 5 SEQ IDNOS:41-51 represent examples of monomeric polypeptide ligand sequences.

Amino acid sequences containing Xaa encompass polypeptides where Xaa isany amino acid.

DETAILED DESCRIPTION OF DRAWINGS

FIGS. 1A-1C show examples of homopolymeric ligands without spacers.

FIGS. 2A-2C show examples of homopolymeric ligands with spacers.

FIGS. 3A-3E show examples of heteropolymeric ligands without spacers.

FIGS. 4A-4F show examples of heteropolymeric ligands with spacers.

FIGS. 5A-5G show examples of ligands and polymeric ligands linked to anoptional epitope tag.

FIGS. 6A-6G show examples of ligands and polymeric ligands linked to anoptional reporter.

FIGS. 7A-7G show examples of ligands and polymeric ligands linked to anoptional localization signal.

FIGS. 8A-8G show examples of ligands and polymeric ligands linked to anoptional localization signal and an optional epitope tag.

FIGS. 9A-9G show examples of gene constructs where ligands andpolyligands are linked to an optional localization signal, an optionalepitope tag, and an optional reporter.

FIGS. 10A-10D show examples of vectors containing ligand geneconstructs.

FIG. 11 shows an example of a sequential cloning process useful forcombinatorial synthesis of polyligands.

FIGS. 12-24 show diagrams of vectors containing gene constructs forligand-beta-galactosidase fusion proteins of the invention.

FIG. 25 shows the average protein concentration ofligand-beta-galactosidase fusion protein in the lysate of transfectedHT-1080 cells.

FIG. 26 shows the results of image analysis of protein dot blot bindingassays of ligand-beta-galactosidase fusion proteins against MEK1 andMEK2 protein targets.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to polypeptide ligands and polyligands for MEK. Anaspect of the invention is to provide novel, modular, inhibitors of MEK(hereafter, the term MEK refers to MEK1 and/or MEK2) 30 activity bymodifying one or more natural substrates or inhibitors by truncationand/or by amino acid substitution. A further aspect of the invention isthe subcellular localization of an MEK inhibitor, ligand, or polyligandby linking to a subcellular localization signal. Various embodiments ofthe MEK ligands and polyligands are represented in SEQ ID NOS:1-51. Morespecifically, the invention relates to ligands, homopolyligands, andheteropolyligands that comprise any one or more of SEQ ID NOS:41-51.Additionally, the invention relates to ligands and polyligandscomprising one or more partial sequences of SEQ ID NOS:37-40 or anyportion thereof. Furthermore, the invention relates to polyligands withat least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequenceidentity to a polyligand comprising one or more of SEQ ID NOS:41-51 orany portion thereof. Furthermore, the invention relates to polyligandswith at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequenceidentity to a polyligand comprising one or more partial sequences of SEQID NOS:37-40.

Polyligands, which can be homopolyligands or heteropolyligands, arechimeric ligands composed of two or more monomeric polypeptide ligands.As used herein, the term chimeric refers to an artificial hybrid orfusion polypeptide containing amino acid sequences from two differentpolypeptides or from different regions of the same polypeptide. Anexample of a monomeric ligand is the polypeptide represented by SEQ IDNO:43, wherein Xaa is any amino acid. SEQ ID NO:43 is a selected partialsequence of wild-type full length SEQ ID NO:39, wherein the amino acidcorresponding to Xaa in the wild-type sequence is a serine, tyrosine, orthreonine phosphorylatable by MEK. An example of a homopolyligand is apolypeptide comprising a dimer or multimer of SEQ ID NO:43, wherein Xaais any amino acid. An example of a heteropolyligand is a polypeptidecomprising SEQ ID NO:51 and one or more of SEQ ID NOS:41-50, wherein Xaais any amino acid. There are numerous ways to combine SEQ ID NOS:41-51into homopolymeric or heteropolymeric ligands. Furthermore, there arenumerous ways to combine additional partial sequences of SEQ IDNOS:37-40 with each other and with SEQ ID NOS:41-51 to make polymericligands.

The polyligands of the invention optionally comprise spacer amino acidsbefore, after, or between monomers. SEQ ID NO:1 is an embodiment of apolyligand of the structure A-S1-B-S2-C-S3-D, wherein A is SEQ ID NO:41,B is SEQ ID NO:42, C is SEQ ID NO:49, and D is SEQ ID NO:43, wherein Xaais alanine or phenylalanine, and wherein S1, S2, and S3 are spacers.This invention intends to capture all combinations of homopolyligandsand heteropolyligands without limitation to the examples given above orbelow. In this description, use of the term “ligand(s)” encompassesmonomeric ligands, polymeric ligands, homopolymeric ligands and/orheteropolymeric ligands.

Monomeric ligands can be categorized into types. One type of monomericligand is a polypeptide where at least a portion of the polypeptide iscapable of being recognized by MEK as a substrate or pseudosubstrate(active site blocker). The portion of the polypeptide capable ofrecognition is termed the recognition motif. In the present invention,recognition motifs can be natural or synthetic. Examples of recognitionmotifs are well known in the art and include, but are not limited to,naturally occurring MEK substrates and pseudosubstrate motifs (SEQ IDNOS:41-48 and partial sequences of SEQ ID NOS:37-39 containing arecognition motif). Another type of monomeric ligand is a polypeptidewhere at least a portion of the polypeptide is capable of associatingwith and inhibiting MEK at a location other than the MEK active site.

A polymeric ligand comprises two or more monomeric ligands linkedtogether to create a chimera.

A homopolymeric ligand is a polymeric ligand where each of the monomericligands is identical in amino acid sequence, except that aphosphorylatable residue may be substituted or modified in one or moreof the monomeric ligands.

A heteropolymeric ligand is a polymeric ligand where some of themonomeric ligands do not have an identical amino acid sequence.

The ligands of the invention are optionally linked to additionalmolecules or amino acids that provide an epitope tag, a reporter, and/ora cellular localization signal. The cellular localization signal targetsthe ligands to a region of a cell. The epitope tag and/or reporterand/or localization signal may be the same molecule. The epitope tagand/or reporter and/or localization signal may also be differentmolecules.

The invention also encompasses polynucleotides comprising a nucleotidesequence encoding ligands, homopolyligands, and heteropolyligands. Thenucleic acids of the invention are optionally linked to additionalnucleotide sequences encoding polypeptides with additional features,such as an epitope tag, a reporter, and/or a cellular localizationsignal. The polynucleotides are optionally flanked by nucleotidesequences comprising restriction endonuclease sites and othernucleotides needed for restriction endonuclese activity. The flankingsequences optionally provide unique cloning sites within a vector andoptionally provide directionality of subsequence cloning. Further, thenucleic acids of the invention are optionally incorporated into vectorpolynucleotides. The ligands, polyligands, and polynucleotides of thisinvention have utility as research tools and/or therapeutics.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to ligands and polyligands that are MEKmodulators. An aspect of the invention is to provide novel, monomericand chimeric, modular inhibitors of MEK activity by modifying one ormore natural substrates or inhibitors by truncation and/or by amino acidsubstitution. A further aspect of the invention is the subcellularlocalization of an MEK inhibitor, ligand, or polyligand by linking to asubcellular localization signal. Various embodiments of ligands andpolyligands are represented in SEQ ID NOS:1-51. Polyligands are chimericligands comprising two or more monomeric polypeptide ligands. An exampleof a monomeric ligand is the polypeptide represented by SEQ ID NO:42,wherein Xaa is any amino acid. SEQ ID NO:42 is a selected partialsequence of parental full length SEQ ID NO:37, wherein the amino acidcorresponding to Xaa in the parent sequence is a serine, tyrosine, orthreonine phosphorylatable by MEK. Another example of a monomeric ligandis the polypeptide represented by SEQ ID NO:49. Another example of amonomeric ligand is the polypeptide represented by SEQ ID NO:46. Each ofSEQ ID NOS:41-51 represents an individual polypeptide ligand inmonomeric form, wherein Xaa is any amino acid. SEQ ID NOS:41-54 areselected examples of partial sequences of SEQ ID NOS:37-40, however,other partial sequences of SEQ ID NOS:37-40 containing a recognitionmotif or binding association motif may also be utilized as monomericligands. Monomeric ligand partial sequences of SEQ ID NOS:37-40 may bewild-type partial sequences. Additionally, monomeric ligand partialsequences of SEQ ID NOS:37-40 may have MEK phosphorylatable amino acidsreplaced by other amino acids. Furthermore, monomeric ligands andpolyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% sequence identity to a ligand comprising an amino acid sequence inone or more of SEQ ID NOS:41-51. Furthermore, monomeric ligands andpolyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%and 99% sequence identity to a partial sequence of SEQ ID NOS:37-40.

An example of a homopolyligand is a polypeptide comprising a dimer ormultimer of SEQ ID NO:50. Another example of a homopolyligand is apolypeptide comprising a dimer or multimer of SEQ ID NO:51. An exampleof a heteropolyligand is a polypeptide comprising SEQ ID NO:41 and oneor more of SEQ ID NOS:42-51, wherein Xaa is any amino acid. There arenumerous ways to combine SEQ ID NOS:41-51 into homopolymeric orheteropolymeric ligands. Furthermore, there are numerous ways to combineadditional partial sequences of SEQ ID NOS:37-40 with each other andwith SEQ ID NOS:41-51 to make polymeric ligands. Polyligands maycomprise any two or more of SEQ ID NOS:41-51, wherein Xaa is any aminoacid. SEQ ID NOS:41-51 are selected examples of partial sequences of SEQID NOS:37-40, however, additional partial sequences, wild-type ormutated, may be utilized to form polyligands. The instant invention isdirected to all possible combinations of homopolyligands andheteropolyligands without limitation.

SEQ ID NOS:41-48 show proteins that contain at least one serine orthreonine residue phosphorylatable by MEK, the positions of which arerepresented by Xaa. Since MEK autophosphorylates, MEK itself is includedas a substrate. SEQ ID NOS:41-48 are partial sequences of SEQ IDNOS:37-39 where, again, the locations of the MEK phosphorylatableresidues are represented by Xaa. In nature, Xaa is, generally speaking,serine, tyrosine, or threonine. In one embodiment of the instantinvention, Xaa can be any amino acid. Ligands where Xaa is serine,tyrosine, or threonine can be used as part of a polyligand; however, inone embodiment, at least one phosphorylatable serine, tyrosine, orthreonine is replaced with another amino acid, such as one of thenaturally occurring amino acids including, alanine, aspartate,asparagine, cysteine, glutamate, glutamine, phenylalanine, glycine,histidine, isoleucine, leucine, lysine, methionine, proline, arginine,valine, or tryptophan. The Xaa may also be a non-naturally occurringamino acid. In another embodiment, the MEK phosphorylatable residue(s)are replaced by alanine. In another embodiment, the MEK phosphorylatableresidue(s) are replaced by phenylalanine. The ligands and polyligands ofthe invention are designed to modulate the endogenous effects of MEK.

In general, ligand monomers based on natural MEK substrates are built byidentifying a putative MEK phosphorylation recognition motif in a MEKsubstrate. Sometimes it is desirable to modify the phosphorylatableresidue to an amino acid other than serine, tyrosine, or threonine.Additional monomers include the MEK recognition motif as well as aminoacids adjacent and contiguous on either side of the MEK recognitionmotif. Monomeric ligands may therefore be any length provided themonomer includes the MEK recognition motif. For example, the monomer maycomprise an MEK recognition motif and at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30-100 or more amino acids adjacent to the recognitionmotif.

For example, in one embodiment, the invention comprises a polypeptideinhibitor of MEK comprising at least one copy of a peptide selected fromthe group consisting of:

a) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 165-203 of SEQ ID NO:39,wherein the amino acid residue corresponding to amino acid residue 185and/or 187 of SEQ ID NO:39 is an amino acid residue other than serine,tyrosine, or threonine;

b) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 169-200 of SEQ ID NO:39,wherein the amino acid residue corresponding to amino acid residue 185and/or 187 of SEQ ID NO:39 is an amino acid residue other than serine,tyrosine, or threonine;

c) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 174-196 of SEQ ID NO:39,wherein the amino acid residue corresponding to amino acid residue 185and/or 187 of SEQ ID NO:39 is an amino acid residue other than serine,tyrosine, or threonine; and

d) a peptide at least 80% identical to a peptide comprising amino acidresidues corresponding to amino acid residues 179-194 of SEQ ID NO:39,wherein the amino acid residue corresponding to amino acid residue 185and/or 187 of SEQ ID NO:39 is an amino acid residue other than serine,tyrosine, or threonine.

As used herein, the terms “correspond(s) to” and “corresponding to,” asthey relate to sequence alignment, are intended to mean enumeratedpositions within a reference protein, e.g., ERK1 (SEQ ID NO:38), andthose positions that align with the positions on the reference protein.Thus, when the 25 amino acid sequence of a subject peptide is alignedwith the amino acid sequence of a reference peptide, e.g., SEQ ID NO:38,the amino acids in the subject peptide sequence that “correspond to”certain enumerated positions of the reference peptide sequence are thosethat align with these positions of the reference peptide sequence, butare not necessarily in these exact numerical positions of the referencesequence. Methods for aligning sequences for determining correspondingamino acids between sequences are described below.

Additional embodiments of the invention include monomers (as describedabove) based on any putative or real substrate for MEK, such assubstrates identified by SEQ ID NOS:37-39. Furthermore, if the substratehas more than one recognition motif, then more than one monomer may beidentified therein.

Another embodiment of the invention is a nucleic acid moleculecomprising a polynucleotide sequence encoding at least one copy of aligand peptide.

Another embodiment of the invention is an isolated polypeptidehomopolyligand, wherein the homopolyligand modulates MEK activity.

Another embodiment of the invention is an isolated polypeptideheteropolyligand, wherein the heteropolyligand modulates MEK activity.

Another embodiment of the invention is a nucleic acid molecule whereinthe polynucleotide sequence 15 encodes one or more copies of one or morepeptide ligands.

Another embodiment of the invention is a nucleic acid molecule whereinthe polynucleotide sequence encodes at least a number of copies of thepeptide selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9 or10.

Another embodiment of the invention is a vector comprising a nucleicacid molecule encoding at least one copy of a ligand or polyligand.

Another embodiment of the invention is a recombinant host cellcomprising a vector comprising a 25 nucleic acid molecule encoding atleast one copy of a ligand or polyligand.

Another embodiment of the invention is a method of inhibiting MEK in acell comprising transfecting a vector comprising a nucleic acid moleculeencoding at least one copy of a ligand or polyligand into a host celland culturing the transfected host cell under conditions suitable toproduce at least one copy of the ligand or polyligand.

The invention also relates to modified inhibitors that are at leastabout 80%, 85%, 90% 95%, 96%, 97%, 98% or 99% identical to a referenceinhibitor. A “modified inhibitor” is used to mean a peptide that can becreated by addition, deletion or substitution of one or more amino acidsin the primary structure (amino acid sequence) of a inhibitor protein orpolypeptide. A “modified recognition motif” is a naturally occurring MEKrecognition motif that has been modified by addition, deletion, orsubstitution of one or more amino acids in the primary structure (aminoacid sequence) of the motif. For example, a modified MEK recognitionmotif may be a motif where the phosphorylatable amino acid has beenmodified to a non-phosphorylatable amino acid. The terms “protein,”“peptide” and “polypeptide” are used interchangeably herein. Thereference inhibitor is not necessarily a wild-type protein or a portionthereof. Thus, the reference inhibitor may be a protein or peptide whosesequence was previously modified over a wild-type protein. The referenceinhibitor may or may not be the wild-type protein from a particularorganism.

A polypeptide having an amino acid sequence at least, for example, about95% “identical” to a reference an amino acid sequence is understood tomean that the amino acid sequence of the polypeptide is identical to thereference sequence except that the amino acid sequence may include up toabout five modifications per each 100 amino acids of the reference aminoacid sequence encoding the reference peptide. In other words, to obtaina peptide having an amino acid sequence at least about 95% identical toa reference amino acid sequence, up to about 5% of the amino acidresidues of the reference sequence may be deleted or substituted withanother amino acid or a number of amino acids up to about 5% of thetotal amino acids in the reference sequence may be inserted into thereference sequence. These modifications of the reference sequence mayoccur at the N-terminus or C-terminus positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among amino acids in the reference sequence or inone or more contiguous groups within the reference sequence.

As used herein, “identity” is a measure of the identity of nucleotidesequences or amino acid sequences compared to a reference nucleotide oramino acid sequence. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. (See, e.g.,Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York (1988); Biocomputing: Informatics And Genome Projects,Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey (1994); von Heinje, G., Sequence Analysis In MolecularBiology, Academic Press (1987); and Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., M Stockton Press, New York (1991)). Whilethere exist several methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math48:1073 (1988)). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego (1994) and Carillo, H. & Lipton, D., Siam J AppliedMath 48:1073 (1988). Computer programs may also contain methods andalgorithms that calculate identity and similarity. Examples of computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP,ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403(1990)) and FASTDB. Examples of methods to determine identity andsimilarity are discussed in Michaels, G. and Garian, R., CurrentProtocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000),which is incorporated by reference. In one embodiment of the presentinvention, the algorithm used to determine identity between two or morepolypeptides is BLASTP.

In another embodiment of the present invention, the algorithm used todetermine identity between two or more polypeptides is FASTDB, which isbased upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990), incorporated by reference). In a FASTDB sequence alignment, thequery and subject sequences are amino sequences. The result of sequencealignment is in percent identity. Parameters that may be used in aFASTDB alignment of amino acid sequences to calculate percent identityinclude, but are not limited to: Matrix=PAM, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or thelength of the subject amino sequence, whichever is shorter.

If the subject sequence is shorter or longer than the query sequencebecause of N-terminus or C-terminus additions or deletions, not becauseof internal additions or deletions, a manual correction can be made,because the FASTDB program does not account for N-terminus andC-terminus truncations or additions of the subject sequence whencalculating percent identity. For subject sequences truncated at bothends, relative to the query sequence, the percent identity is correctedby calculating the number of amino acids of the query sequence that areN- and C-terminus to the reference sequence that are notmatched/aligned, as a percent of the total amino acids of the querysequence. The results of the FASTDB sequence alignment determinematching/alignment. The alignment percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score can be used for the purposes of determining howalignments “correspond” to each other, as well as percentage identity.Residues of the query (subject) sequences or the reference sequence thatextend past the N- or C-termini of the reference or subject sequence,respectively, may be considered for the purposes of manually adjustingthe percent identity score. That is, residues that are notmatched/aligned with the N- or C-termini of the comparison sequence maybe counted when manually adjusting the percent identity score oralignment numbering.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue reference sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a match/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 reference sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery.

In this case the percent identity calculated by FASTDB is not manuallycorrected.

The polyligands of the invention optionally comprise spacer amino acidsbefore, after, or between monomers. The length and composition of thespacer may vary. An example of a spacer is glycine, alanine,polyglycine, or polyalanine. Specific examples of spacers used betweenmonomers in SEQ ID NO:1 are the five amino acid spacers PGAAG and PAGGA.In the instance of SEQ ID NO:1, the proline-containing spacers areintended to break secondary structure. Spacer amino acids may be anyamino acid and are not limited to these alanine, glycine, andproline-containing examples. The instant invention is directed to allcombinations of homopolyligands and heteropolyligands, with or withoutspacers, and without limitation to the examples given above or below.

The ligands and polyligands of the invention are optionally linked toadditional molecules or amino acids that provide an epitope tag, areporter, and/or localize the ligand to a region of a cell (See FIGS.5A-5G, FIGS. 6A-6G, FIGS. 7A-7G, and FIGS. 8A-8G).

Non-limiting examples of epitope tags are FLAG™, HA (hemagluttinin),c-Myc and His6. Non-limiting examples of reporters are alkalinephosphatase, galactosidase, peroxidase, luciferase and fluorescentproteins. Non-limiting examples of cellular localizations aresarcoplamic reticulum, endoplasmic reticulum, mitochondria, golgiapparatus, nucleus, plasma membrane, apical membrane, and basolateralmembrane. The epitopes, reporters and localization signals are given byway of example and without limitation. The epitope tag, reporter and/orlocalization signal may be the same molecule. The epitope tag, reporterand/or localization signal may also be different molecules.

Ligands and polyligands and optional amino acids linked thereto can besynthesized chemically or recombinantly using techniques known in theart. Chemical synthesis techniques include but are not limited topeptide synthesis which is often performed using an automated peptidesynthesizer. Peptides can also be synthesized utilizing non-automatedpeptide synthesis methods known in the art. Recombinant techniquesinclude insertion of ligand-encoding nucleic acids into expressionvectors, wherein nucleic acid expression products are synthesized usingcellular factors and processes.

Linkage of a cellular localization signal, epitope tag, or reporter to aligand or polyligand can include covalent or enzymatic linkage to theligand. When the localization signal comprises material other than apolypeptide, such as a lipid or carbohydrate, a chemical reaction tolink molecules may be utilized.

Additionally, non-standard amino acids and amino acids modified withlipids, carbohydrates, phosphate or other molecules may be used asprecursors to peptide synthesis.

The ligands of the invention have therapeutic utility with or withoutlocalization signals. However, ligands linked to localization signalshave utility as subcellular tools or therapeutics. For example, 25ligands depicted generically in FIGS. 7A-7G represent ligands withutility as subcellular tools or therapeutics. MEK ligand-containing geneconstructs are also delivered via gene therapy. FIGS. 10B and 10C depictembodiments of gene therapy vectors for delivering and controllingpolypeptide expression in vivo. Polynucleotide sequences linked to thegene construct in FIGS. 10B and 10C include genome integration domainsto facilitate integration of the transgene into a viral genome and/orhost genome.

FIG. 10A shows a vector containing an MEK ligand gene construct, whereinthe ligand gene construct is releasable from the vector as a unit usefulfor generating transgenic animals. For example, the ligand geneconstruct, or transgene, is released from the vector backbone byrestriction endonuclease digestion. The released transgene is theninjected into pronuclei of fertilized mouse eggs; or the transgene isused to transform embryonic stem cells. The vector containing a ligandgene construct of FIG. 10A is also useful for transient transfection ofthe transgene, wherein the promoter and codons of the transgene areoptimized for the host organism. The vector containing a ligand geneconstruct of FIG. 10A is also useful for recombinant expression ofpolypeptides in fermentable organisms adaptable for small or large scaleproduction, wherein the promoter and codons of the transgene areoptimized for the fermentation host organism.

FIG. 10D shows a vector containing an MEK ligand gene construct usefulfor generating stable cell lines.

The invention also encompasses polynucleotides comprising nucleotidesequences encoding ligands, homopolyligands, and heteropolyligands. Thepolynucleotides of the invention are optionally linked to additionalnucleotide sequences encoding epitopes, reporters and/or localizationsignals. Further, the nucleic acids of the invention are optionallyincorporated into vector polynucleotides. The polynucleotides areoptionally flanked by nucleotide sequences comprising restrictionendonuclease sites and other nucleotides needed for restrictionendonuclese activity. The flanking sequences optionally provide cloningsites within a vector. The restriction sites can include, but are notlimited to, any of the commonly used sites in most commerciallyavailable cloning vectors. Examples of such sites are those recognizedby BamHI, ClaI, EcoRI, EcoRV, SpeI, AflII, NdeI, NheI, XbaI, XhoI, SphI,NaeI, SexAI, HindIII, HpaI, and PstT restriction endonucleases. Sitesfor cleavage by other restriction enzymes, including homingendonucleases, are also used for this purpose. The polynucleotideflanking sequences also optionally provide directionality of partialsequence cloning. It is preferred that 5′ and 3′ restrictionendonuclease sites differ from each other so that double-stranded DNAcan be directionally cloned into corresponding complementary sites of acloning vector.

Ligands and polyligands with or without localization signals, epitopesor reporters are alternatively synthesized by recombinant techniques.Polynucleotide expression constructs are made containing desiredcomponents and inserted into an expression vector. The expression vectoris then transfected into cells and the polypeptide products areexpressed and isolated. Ligands made according to recombinant DNAtechniques have utility as research tools and/or therapeutics.

The following is an example of how polynucleotides encoding ligands andpolyligands are produced. Complimentary oligonucleotides encoding theligands and flanking sequences are synthesized and annealled. Theresulting double-stranded DNA molecule is inserted into a cloning vectorusing techniques known in the art. When the ligands and polyligands areplaced in-frame adjacent to sequences within a transgenic gene constructthat is translated into a protein product, they form part of a fusionprotein when expressed in cells or transgenic animals.

Another embodiment of the invention relates to selective control oftransgene expression in a desired cell or organism. The promotor portionof the recombinant gene can be a constitutive promotor, anon-constitutive promotor, a tissue-specific promotor (constitutive ornon-constitutive) or a selectively controlled promotor. Differentselectively controlled promotors are controlled by different mechanisms.For example, RheoSwitch is an inducible promotor system available fromNew England Biolabs.

Temperature sensitive promotors can also be used to increase or decreasegene expression. An embodiment of the invention comprises a ligand orpolyligand gene construct whose expression is controlled by an induciblepromotor. In one embodiment, the inducible promotor is tetracyclinecontrollable.

Polyligands are modular in nature. An aspect of the instant invention isthe combinatorial modularity of the disclosed polyligands. Anotheraspect of the invention are methods of making these modular polyligandseasily and conveniently. In this regard, an embodiment of the inventioncomprises methods of modular partial sequence cloning of geneticexpression components. When the ligands, homopolyligands,heteropolyligands and optional amino acid expression components aresynthesized recombinantly, one can consider each clonable element as amodule. For speed and convenience of cloning, it is desirable to makemodular elements that are compatible at cohesive ends and are easy toinsert and clone sequentially. This is accomplished by exploiting thenatural properties of restriction endonuclease site recognition andcleavage. One aspect of the invention encompasses module flankingsequences that, at one end of the module, are utilized for restrictionenzyme digestion once, and at the other end, utilized for restrictionenzyme digestion as many times as desired. In other words, a restrictionsite at one end of the module is utilized and destroyed in order toeffect sequential cloning of modular elements. An example of restrictionsites flanking a coding region module are sequences recognized by therestriction enzymes NgoM IV and Cla I; or Xma I and Cla I. Cutting afirst circular DNA with NgoM IV and Cla I to yield linear DNA with a 5′NgoM IV overhang and a 3′ Cla I overhang; and cutting a second circularDNA with Xma I and Cla I to yield linear DNA with a 5′ Cla I overhangand a 3′ Xma I overhang generates first and second DNA fragments withcompatible cohesive ends. When these first and second DNA fragments aremixed together, annealed, and ligated to form a third circular DNAfragment, the NgoM IV site that was in the first DNA and the Xma I sitethat was in the second DNA are destroyed in the third circular DNA. Nowthis vestigial region of DNA is protected from further Xma I or NgoM IVdigestion, but flanking sequences remaining in the third circular DNAstill contain intact 5′ NgoM IV and 3′ Cla I sites. This process can berepeated numerous times to achieve directional, sequential, modularcloning events. Restriction sites recognized by NgoM IV, Xma I, and ClaI endonucleases represent a group of sites that permit sequentialcloning when used as flanking sequences.

Another way to assemble coding region modules directionally andsequentially employs linear DNA in addition to circular DNA. Forexample, like the sequential cloning process described above,restriction sites flanking a coding region module are sequencesrecognized by the restriction enzymes NgoM IV and Cla I; or Xma I andCla I. A first circular DNA is cut with NgoM IV and Cla I to yieldlinear DNA with a 5′ NgoM IV overhang and a 3′ Cla I overhang. A secondlinear double-stranded DNA is generated by PCR amplification or bysynthesizing and annealing complimentary oligonucleotides. The secondlinear DNA has 5′ Cla I overhang and a 3′ Xma I overhang, which arecompatible cohesive ends with the first DNA linearized. When these firstand second DNA fragments are mixed together, annealed, and ligated toform a third circular DNA fragment, the NgoM IV site that was in thefirst DNA and the Xma I site that was in the second DNA are destroyed inthe third circular DNA. Flanking sequences remaining in the thirdcircular DNA still contain intact 5′ NgoM IV and 3′ Cla I sites. Thisprocess can be repeated numerous times to achieve directional,sequential, modular cloning events. Restriction sites recognized by NgoMIV, Xma I, and Cla I endonucleases represent a group of sites thatpermit sequential cloning when used as flanking sequences. This processis depicted in FIG. 11.

One of ordinary skill in the art recognizes that other restriction sitegroups can accomplish sequential, directional cloning as describedherein. Preferred criteria for restriction endonuclease selection areselecting a pair of endonucleases that generate compatible cohesive endsbut whose sites are destroyed upon ligation with each other. Anothercriteria is to select a third endouclease site that does not generatesticky ends compatible with either of the first two. When such criteriaare utilized as a system for sequential, directional cloning, ligands,polyligands and other coding regions or expression components can becombinatorially assembled as desired. The same sequential process can beutilzed 5 for epitope, reporter, and/or localization signals.

Polyligands and methods of making polyligands that modulate MEK activityare disclosed. Therapeutics include delivery of purified ligand orpolyligand with or without a localization signal to a cell.Alternatively, ligands and polyligands with or without a localizationsignals are delivered via adenovirus, lentivirus, adeno-associatedvirus, or other viral constructs that express protein product in a cell.

Example 1

A polypeptide comprising a heteropolyligand, an endoplasmic reticulumcellular localization signal, and a His6 epitope is synthesized.Examples of such polypeptides are generically represented by FIGS. 8A,8B, 8D, 8E and 8F. The polypeptide is synthesized on an automatedpeptide synthesizer or is recombinantly expressed and purified. Purifiedpolypeptide is solubilized in media and added to cells. The polypeptideis endocytosed by the cells, and transported to the endoplasmicreticulum. Verification is performed by immunohistochemical stainingusing an anti-His6 antibody.

Example 2

A transgene is constructed using a cytomegalovirus (CMV) promoter todirect expression of a fusion protein comprising SEQ ID NO:49, SEQ IDNO:48, SEQ ID NO:41, wherein Xaa is alaninc (POLYLIGAND), greenfluorescent protein (REPORTER), and a plasma membrane localizationsignal (LOCALIZATION SIGNAL). Such a transgene is genericallyrepresented by FIG. 9C. The transgene is transfected into cells fortransient expression. Verification of expression and location isperformed by visualization of green fluorescent protein by confocalmicroscopy.

Example 3

A transgene construct is built to produce a protein product withexpression driven by a tissue-specific promoter. The transgene comprisesa synthetic gene expression unit engineered to encode three domains.Each of these three domains is synthesized as a pair of complimentarypolynucleotides that are annealed in solution, ligated and inserted intoa vector. Starting at the amino-terminus, the three domains in theexpression unit are nucleotide sequences that encode an MEK ligand, aFLAG™ epitope, and a nuclear localization signal. The MEK ligand is amonomeric ligand, homopolymeric ligand or heteropolymeric ligand asdescribed herein. Nucleotide sequences encoding a FLAG™ epitope areplaced downstream of nucleotide sequences encoding the MEK ligand.Finally, nucleotide sequences encoding the localization signal areplaced downstream of those encoding the FLAG™ epitope. The assembledgene expression unit is subsequently subcloned into an expressionvector, such as that shown in FIG. 10A, and used to transientlytransfect cells. Verification is performed by immunohistochemicalstaining using an anti-FLAG™ antibody.

Example 4

Modulation of MEK cellular function by subcellularly localized MEKpolyligand is illustrated. A transgene construct containing nucleicacids that encode a polyligand fusion protein, epitope, and endoplasmicreticulum localization signal is made. The expression unit containsnucleotides that encode SEQ ID NO:25 (POLYLIGAND), a c-Myc epitope(EPITOPE), and a nuclear localization signal (LOCALIZATION SIGNAL). Thisexpression unit is subsequently subcloned into a vector between aEF1alpha promoter and an SV40 polyadenylation signal. The completedtransgenecontaining expression vector is then used to transfect cells.Inhibition of MEK activity is demonstrated by measuring phosphorylationof endogenous substrates against controls and/or observing phenotypes.

Example 5

Ligand function and localization is demonstrated in vivo by making atransgene construct used to generate mice expressing a ligand fusionprotein targeted to the nucleus. The transgene construct is showngenerically in FIG. 10B. The expression unit contains nucleotides thatencode a tetramer of SEQ ID NO:33, a hemagluttinin epitope, and anuclear localization signal. This expression unit is subsequentlysubcloned into a vector between nucleotide sequences including aninducible promoter and an SV40 polyadenylation signal. The completedtransgene is then injected into pronuclei of fertilized mouse oocytes.The resultant pups are screened for the presence of the transgene byPCR. Transgenic founder mice are bred with wild-type mice. Heterozygoustransgenic animals from at least the third generation are used for thefollowing tests, with their non-transgenic littermates serving ascontrols.

Test 1: Southern blotting analysis is performed to determine the copynumber. Southern blots are hybridized with a radio-labeled probegenerated from a fragment of the transgene. The probe detects bandscontaining DNA from transgenic mice, but does not detect bandscontaining DNA from non-transgenic mice. Intensities of the transgenicmice bands are measured and compared with the transgene plasmid controlbands to estimate copy number. This demonstrates that mice in Example 5harbor the transgene in their genomes.

Test 2: Tissue homogenates are prepared for Western blot analysis. Thisexperiment demonstrates the transgene is expressed in tissues oftransgenic mice because hemagluttinin epitope is detected in transgenichomogenates but not in non-transgenic homogenates.

Test 3: Function is assessed by phenotypic observation or analysisagainst controls after induction of expression.

These examples demonstrate delivery of ligands to a localized region ofa cell for therapeutic or experimental purposes. The purifiedpolypeptide ligands can be formulated for oral or parenteraladministration, topical administration, or in tablet, capsule, or liquidform, intranasal or inhaled aerosol, subcutaneous, intramuscular,intraperitoneal, or other injection; intravenous instillation; or anyother routes of administration. Furthermore, the nucleotide sequencesencoding the ligands permit incorporation into a vector designed todeliver and express a gene product in a cell. Such vectors includeplasmids, cosmids, artificial chromosomes, and modified viruses.Delivery to eukaryotic cells can be accomplished in vivo or ex vivo. Exvivo delivery methods include isolation of the intended recipient'scells or donor cells and delivery of the vector to those cells, followedby treatment of the recipient with the cells.

Example 6

Fusion proteins were constructed with ligands of the present inventionfused to beta-galactosidase.

FIG. 12 represents a vector that comprises a polynucleotide that encodesthe polyligand of SEQ ID NO:33. In the vector of FIG. 12, thepolynucleotide encoding the ligand of SEQ ID NO:33 is linked to apolynucleotide encoding beta-galactosidase to create a ligand fusionprotein coding sequence.

FIG. 13 represents a vector that comprises the polynucleotide of SEQ IDNO:2, which encodes the ligand of SEQ ID NO:1. In the vector of FIG. 13,the polynucleotide of SEQ ID NO:2 is linked to a polynucleotide encodingbeta-galactosidase to create a ligand fusion protein coding sequence.

FIG. 14 represents a vector that comprises the polynucleotide of SEQ IDNO:6, which encodes the ligand of SEQ ID NO:5. In the vector of FIG. 14,the polynucleotide of SEQ ID NO:6 is linked to a polynucleotide encodingbeta-galactosidase to create a ligand fusion protein coding sequence.

FIG. 15 represents a vector that comprises the polynucleotide of SEQ IDNO:10, which encodes the ligand of SEQ ID NO:9. In the vector of FIG.15, the polynucleotide of SEQ ID NO:10 is linked to a polynucleotideencoding beta-galactosidase to create a ligand fusion protein codingsequence.

FIG. 16 represents a vector that comprises the polynucleotide of SEQ IDNO:14, which encodes the ligand of SEQ ID NO:13. In the vector of FIG.16, the polynucleotide of SEQ ID NO:14 is linked to a polynucleotideencoding beta-galactosidase to create a ligand fusion protein codingsequence.

FIG. 17 represents a vector that comprises the polynucleotide of SEQ IDNO:18, which encodes the ligand of SEQ ID NO:17. In the vector of FIG.17, the polynucleotide of SEQ ID NO:18 is linked to a polynucleotideencoding beta-galactosidase to create a ligand fusion protein codingsequence.

FIG. 18 represents a vector that comprises the polynucleotide of SEQ IDNO:22, which encodes the ligand of SEQ ID NO:21. In the vector of FIG.18, the polynucleotide of SEQ ID NO:22 is linked to a polynucleotideencoding beta-galactosidase to create a ligand fusion protein codingsequence.

FIG. 19 represents a vector that comprises SEQ ID NO:26, which encodesthe ligand of SEQ ID NO:25. In the vector of FIG. 19, the polynucleotideof SEQ ID NO:26 is linked to a polynucleotide encodingbeta-galactosidase to create a ligand fusion protein coding sequence.

FIG. 20 represents another vector that comprises SEQ ID NO:26, whichencodes the ligand of SEQ ID NO:25. In the vector of FIG. 20, thepolynucleotide of SEQ ID NO:26 is linked to a polynucleotide encodingbeta-galactosidase to create a ligand fusion protein coding sequence.

FIG. 21 represents a vector that comprises SEQ ID NO:30, which encodesthe ligand of SEQ ID NO:29. In the vector of FIG. 21, the polynucleotideof SEQ ID NO:30 is linked to a polynucleotide encodingbeta-galactosidase to create a ligand fusion protein coding sequence.

FIG. 22 represents another vector that comprises SEQ ID NO:30, whichencodes the ligand of SEQ ID NO:29. In the vector of FIG. 22, thepolynucleotide of SEQ ID NO:30 is linked to a polynucleotide encodingbeta-galactosidase to create a fusion protein coding sequence.

FIG. 23 represents a vector that comprises SEQ ID NO:34, which encodesthe ligand of SEQ ID NO:33. In the vector of FIG. 23, the polynucleotideof SEQ ID NO:34 is linked to a polynucleotide encodingbeta-galactosidase to create a ligand fusion protein coding sequence.

FIG. 24 represents another vector that comprises SEQ ID NO:34, whichencodes the ligand of SEQ ID NO:33. In the vector of FIG. 24, thepolynucleotide of SEQ ID NO:34 is linked to a polynucleotide encodingbeta-galactosidase to create a ligand fusion protein coding sequence.

The vectors of FIGS. 12-24 were transfected into the mammalian cell lineHT1080. A vector comprising a polynucleotide encoding the MEK substrateERK was also transfected in HT1080 cells. Transfections were performedusing Fugene6 reagent—purchased from Roche (Basel,Switzerland)—according to the manufacture specifications. Briefly, thecells were seeded into 6-well plates at a density of 300,000 per well in2 ml of DMEM with 10% of FBS. 24 hours later, the transfection complexesper each well were prepared by mixing of 1 ug of plasmid DNA with 3 ulof Fugene6 which resulted in 100 ul of the DNA/lipid complex dissolvedin the serum free DMEM. After 30 minutes of incubation allowing toproperly form the complex 100 ul of the mixture was added to each wellof cells growing in 2 ml of medium. Cells were exposed to the DNA/lipidcomplexes for 24 hours and subsequently lyzed for the RNA and proteinanalysis.

FIG. 25 shows the results of protein analyses of the lysate for ligandfusion proteins encoded by the vectors of FIGS. 12-18 and FIGS. 20, 22and 24. The ligand fusion proteins were quantified using thebeta-galactosidase ELISA kit from Roche (#11539426001) according to kitprotocol. Values represent the average of two replicates, except forVVN-40647 which represents the value of 1 replicate.

The lysates containing ligand fusion proteins were assayed in a proteindot-blot binding assay for the detection of binding to MEK1 protein orMEK2 protein using the following protocol:

-   -   1. Soak a nitrocellulose membrane in TBS for 5 minutes.    -   2. Place pre-soaked nitrocellulose membrane into dot-blot        apparatus, apply vacuum and seal apparatus by tightening screws.    -   3. Re-hydrate nitrocellulose by adding 100 μL of TBS to each        well. Briefly apply vacuum but DO NOT completely dry the wells.    -   4. Confirm flow valve/vacuum chamber of the dot-blot apparatus        is open to air and fill wells with 100 μL of a 0.5 ng/μL        solution of either MEK1 or MEK2 target protein for a final assay        amount of 50 ng of target protein per well.    -   5. Allow the target protein to filter through the membrane by        gravity flow for 40 minutes at room temperature before drawing        the remaining liquid through the membrane by vacuum filtration.    -   6. Block wells by adding 300 μL of 5% non-fat dried milk in TBS        for 1 hour at room temperature.    -   7. Carefully aspirate the 5% blocking solution from each well.    -   8. Wash each well once by adding 100 μL of TBS. Pull the TBS        through the membrane by vacuum filtration.    -   9. Add 100 μL of a 0.1 ng/μL solution of each of the MEK        inhibitor lysates to by tested to one well containing MEK1        protein and one well containing MEK2 protein for a final assay        amount of 10 ng of inhibitor per well. Incubate inhibitor        lysates with MEK1/2 target protein for 40 minutes at room        temperature before drawing the remaining lysate through the        membrane by vacuum filtration.    -   10. Wash each well by adding 100 μL 1% SDS in TBS. Pull the 1%        SDS in TBS through the membrane by vacuum filtration. Repeat        this wash step two additional times for a total of three washes.    -   11. Apply vacuum and mark the membrane with a pen or pencil (so        that membrane can be re-aligned after removal from the        apparatus).    -   12. Turn off vacuum and remove the membrane from the apparatus.    -   13. Place membrane in a Petri dish (or equivalent vessel) and        wash with 1% SDS in TBS for 5 minutes with gentle agitation (add        enough 1% SDS in TBS to cover the entire membrane). Repeat this        wash step four additional times for a total of five washes.    -   14. Wash membrane once in TBS (as described in step 13) to        remove excess SDS detergent from the membrane.    -   15. Return membrane to the dot-blot apparatus as described in        step 2.    -   16. Add 100 μL of βeta-Glo beta-galactosidase substrate to each        well and incubate for 30 minutes at room temperature.    -   17. Vacuum filter βeta-Glo substrate through the membrane,        remove the membrane from the apparatus and expose the membrane        for 15 minutes in the FluorChem imager set for chemiluminescent        detection.

The materials used in this assay were:

-   -   1. Dot-blot apparatus (Bio-Rad or equivalent)    -   2. Tris (Sigma, #252859 or equivalent)    -   3. SDS (ICN #811034 or equivalent)    -   4. NaCl (EMD #7647-14-5 or equivalent)    -   5. βeta-Glo Assay Kit (Promega #E4740)    -   6. Mek1 (Cell Signaling #M02-10G-10 or equivalent)    -   7. Mek2 (Cell Signaling #M03-10G-10 or equivalent)    -   8. FluorChem imager (Alpha Innotech or equivalent)

The image data was then quantified using the following protocol:

-   -   1. Open the dot blot image in the software application, ImageJ.    -   2. Use the rectangular selection tool to outline the first row.    -   3. Select Mark First Lane in the Special menu.    -   4. Move the rectangular selection (by clicking inside it and        dragging) and outline (using Mark Next Lane) each of the other        lanes in succession.    -   5. Use Plot Lanes to generate the lane profile plots.    -   6. Use the line drawing tool to draw base lines and drop lines        so that each peak defines a closed area as shown above.    -   7. Measure the areas of the peaks by clicking inside each one in        succession with the wand tool.    -   8. The data file with the peak measurements (dot intensity        values) can then be saved as an Microsoft excel file and        normalized and graphed.

FIG. 26 represents the results of the image analysis of the protein dotblot binding assay, which shows that several of the fusion proteinsexhibited binding activity against MEK1 and/or MEK 2.

Disclosed are ligands and polyligands that modulate MEK activity andmethods of making and using these ligands. The ligands and polyligandsare synthesized chemically or recombinantly and are utilized as researchtools or as therapeutics. The invention includes linking the ligands andpolyligands to cellular localization signals for subcellulartherapeutics.

1.-26. (canceled)
 27. A polynucleotide encoding a polypeptide polyligandcomprising one or more copies of a polypeptide monomer selected from thegroup consisting of an amino acid sequence at least 80% identical to anyone of SEQ ID NOS: 41-51, wherein the polypeptide polyligand inhibitsMEK activity.
 28. The polynucleotide of claim 27, wherein thepolypeptide polyligand is a polypeptide homopolyligand.
 29. Thepolynucleotide of claim 27, wherein the polypeptide polyligand is apolypeptide heteropolyligand.
 30. The polynucleotide of claim 27,wherein Xaa in any one of SEQ ID NOS: 41-51 is any amino acid.
 31. Thepolynucleotide of claim 27, wherein Xaa in any one of SEQ ID NOS: 41-51an amino acid other than serine, tyrosine or threonine.
 32. Thepolynucleotide of claim 27, wherein the polypeptide polyligand is linkedto a subcellular localization signal, a reporter, and/or an epitope tag.33. The polynucleotide of claim 27, wherein the polynucleotide isisolated.
 34. A vector comprising the polynucleotide of claim
 27. 35. Ahost cell comprising the vector of claim
 34. 36. A method of inhibitingMEK in a host cell, the method comprising transfecting the vector ofclaim 34 into a host cell, and culturing the host cell under conditionssuitable to produce at least one copy of the polypeptide polyligand. 37.The polynucleotide of claim 27, wherein the polypeptide monomer is atleast 85% identical to any one of SEQ ID NOS: 41-51.
 38. Thepolynucleotide of claim 27, wherein the polypeptide monomer is at least90% identical to any one of SEQ ID NOS: 41-51.
 39. The polynucleotide ofclaim 27, wherein the polypeptide monomer is at least at least 95%identical to any one of SEQ ID NOS: 41-51.
 40. The polynucleotide ofclaim 27, wherein the polypeptide monomer is at least at least 96%identical to any one of SEQ ID NOS: 41-51.
 41. The polynucleotide ofclaim 27, wherein the polypeptide monomer is at least at least 97%identical to any one of SEQ ID NOS: 41-51.
 42. The polynucleotide ofclaim 27, wherein the polypeptide monomer is at least at least 98%identical to any one of SEQ ID NOS: 41-51.
 43. The polynucleotide ofclaim 27, wherein the polypeptide monomer is at least 99% identical toany one of SEQ ID NOS: 41-51.
 44. The polynucleotide of claim 27,wherein the polypeptide monomer is one of SEQ ID NOS: 41-51.